Thin Filament Regulatory Units Research Articles

The spatial distribution of thin filament activation influences force development and myosin activity in computational models of muscle contraction

Striated muscle contraction is initiated by Ca2+ binding to, and activating, thin filament regulatory units (RU) within the sarcomere, which then allows myosin cross-bridges from the opposing thick filament to bind actin and generate force. The amount of overlap between the filaments dictates how many potential cross-bridges are capable of binding, and thus how force is generated by the sarcomere. Myopathies and atrophy can impair muscle function by limiting cross-bridge interactions between the filaments, which can occur when the length of the thin filament is reduced or when RU function is disrupted. To investigate how variations in thin filament length and RU density affect ensemble cross-bridge behavior and force production, we simulated muscle contraction using a spatially explicit computational model of the half-sarcomere. Thin filament RUs were disabled either uniformly from the pointed end of the filament (to model shorter thin filament length) or randomly throughout the length of the half-sarcomere. Both uniform and random RU ‘knockout’ schemes decreased overall force generation during maximal and submaximal activation. The random knockout scheme also led to decreased calcium sensitivity and cooperativity of the force-pCa relationship. We also found that the rate of force development slowed with the random RU knockout, compared to the uniform RU knockout or conditions of normal RU activation. These findings imply that the relationship between RU density and force production within the sarcomere involves more complex coordination than simply the raw number of RUs available for myosin cross-bridge binding, and that the spatial pattern in which activatable RU are distributed throughout the sarcomere influences the dynamics of force production.

Potential impacts of the cardiac troponin I mobile domain on myofilament activation and relaxation

The cardiac thin filament is regulated in a Ca2+-dependent manner through conformational changes of troponin and tropomyosin (Tm). It has been generally understood that under conditions of low Ca2+ the inhibitory peptide domain (IP) of troponin I (TnI) binds to actin and holds Tm over the myosin binding sites on actin to prevent crossbridge formation. More recently, evidence that the C-terminal mobile domain (MD) of TnI also binds actin has made for a more complex scenario. This study uses a computational model to investigate the consequences of assuming that TnI regulates Tm movement via two actin-binding domains rather than one. First, a 16-state model of the cardiac thin filament regulatory unit was created with TnI-IP as the sole regulatory domain. Expansion of this to include TnI-MD formed a 24-state model. Comparison of these models showed that assumption of a second actin-binding site allows the individual domains to have a lower affinity for actin than would be required for IP acting alone. Indeed, setting actin affinities of the IP and MD to 25% of that assumed for the IP in the single-site model was sufficient to achieve precisely the same degree of Ca2+ regulation. We also tested the 24-state model's ability to represent steady-state experimental data in the case of disruption of either the IP or MD. We were able to capture qualitative changes in several properties that matched what was seen in the experimental data. Lastly, simulations were run to examine the effect of disruption of the IP or MD on twitch dynamics. Our results suggest that both domains are required to keep diastolic cross-bridge activity to a minimum and accelerate myofilament relaxation. Overall, our analyses support a paradigm in which two domains of TnI bind with moderate affinity to actin, working in tandem to complete Ca2+-dependent regulation of the thin filament.

A comprehensive guide to genetic variants and post-translational modifications of cardiac troponin C.

Familial cardiomyopathy is an inherited disease that affectsthe structure and function of heartmuscle and has an extreme range of phenotypes. Among the millions of affected individuals, patients with hypertrophic (HCM), dilated (DCM), or left ventricular non-compaction (LVNC) cardiomyopathy can experience morphologic changes of the heart which lead to sudden death in the most detrimental cases. TNNC1, the gene that codes for cardiac troponin C (cTnC), is a sarcomere gene associated with cardiomyopathies in which probands exhibit young age of presentation and high death, transplant or ventricular fibrillation events relative to TNNT2 and TNNI3 probands. Using GnomAD, ClinVar, UniProt and PhosphoSitePlus databases and published literature, an extensive list to date of identified genetic variants in TNNC1 and post-translational modifications (PTMs) in cTnC was compiled. Additionally, a recent cryo-EM structure of the cardiac thin filament regulatory unit was used to localize each functionally studied amino acid variant and each PTM (acetylation, glycation, s-nitrosylation, phosphorylation) in the structure of cTnC. TNNC1 has a large number of variants (> 100) relative to other genes of the same transcript size. Surprisingly, the mapped variant amino acids and PTMs are distributed throughout the cTnC structure. While many cardiomyopathy-associated variants are localized in α-helical regions of cTnC, this was not statistically significant χ2 (p = 0.72). Exploring the variants in TNNC1 and PTMs of cTnC in the contexts of cardiomyopathy association, physiological modulation and potential non-canonical roles provides insights into the normal function of cTnC along with the many facets of TNNC1 as a cardiomyopathic gene.

Turning the Azimuthal Motions of Adjacent Tropomyosins into a Coupled N-body Problem in a Brownian Model of Cardiac Thin Filament Activation

During thin filament activation process, tropomyosin (Tm) oscillates over the surface of actin filament in the azimuthal direction to regulate the access of myosin head to actin binding sites. This motion is characterized by the existence of three distinct equilibrium positions, namely; blocked (B), closed (C), and open (M) regulatory states. The exact dynamics and energy landscape that governs Tm motions between these conformational states is not yet fully understood. In this study, we developed a Brownian flashing ratchet model to mathematically describe Tm motions over the surface of actin filament. The model approximates the Tm flexible chain by a system of discrete and coupled Brownian bodies. In other words, the thin filament regulatory units (RUs) are prescribed by a set of N-body stochastic ODEs that govern the azimuthal motion of adjacent tropomyosins. The model explicitly accounts for spatial interactions among nearest neighbor RUs, which were thought to appear from structural coupling of adjacent tropomyosins. This coupling is achieved by assigning each Brownian Tm-body with a local multi-well potential. The energetic barrier, asymmetry, and bias of each individual energy landscape are hypothesized to be influenced by the Tm status of its neighbors. This coupling approach is anticipated to be sufficient to produce observed cooperativity in both force-Ca2+ relationship and twitch dynamic. The present analysis is expected to perform better than many of current phenomenological myofilament models which were mostly derived based on the classical mean-field approximation. Additionally, this analysis draws a more detailed molecular connection between Tm dynamics and sarcomeric twitch production. Therefore, the Brownian ratchet modeling methodology of Tm motions during thin filament activation may offer an enhanced mechanistic approach to describe cardiac muscle contraction.

Predicting Effects of Tropomyosin Mutations on Cardiac Muscle Contraction through Myofilament Modeling.

Point mutations to the human gene TPM1 have been implicated in the development of both hypertrophic and dilated cardiomyopathies. Such observations have led to studies investigating the link between single residue changes and the biophysical behavior of the tropomyosin molecule. However, the degree to which these molecular perturbations explain the performance of intact sarcomeres containing mutant tropomyosin remains uncertain. Here, we present a modeling approach that integrates various aspects of tropomyosin's molecular properties into a cohesive paradigm representing their impact on muscle function. In particular, we considered the effects of tropomyosin mutations on (1) persistence length, (2) equilibrium between thin filament blocked and closed regulatory states, and (3) the crossbridge duty cycle. After demonstrating the ability of the new model to capture Ca-dependent myofilament responses during both dynamic and steady-state activation, we used it to capture the effects of hypertrophic cardiomyopathy (HCM) related E180G and D175N mutations on skinned myofiber mechanics. Our analysis indicates that the fiber-level effects of the two mutations can be accurately described by a combination of changes to the three tropomyosin properties represented in the model. Subsequently, we used the model to predict mutation effects on muscle twitch. Both mutations led to increased twitch contractility as a consequence of diminished cooperative inhibition between thin filament regulatory units. Overall, simulations suggest that a common twitch phenotype for HCM-linked tropomyosin mutations includes both increased contractility and elevated diastolic tension.

Modeling Predicts Non-Monotonic Dependence of Myofilament Ca2+ Sensitivity on Tropomyosin Stiffness

The cooperative Ca2+ activation of the thin filament in cardiac muscle plays a vital role in the contraction and relaxation of the heart. Nearest-neighbor interactions between thin filament regulatory units (RUs) are widely considered the principal mechanism of cooperativity, but descriptions of these interactions vary. Previous models have assumed that interactions can be partitioned into distinct categories, such as RU-RU, RU-crossbridge (XB), and XB-XB cooperativity. Under these schemes, the strength of each type of cooperativity is described by independently adjusted parameters. While such models have produced useful insight and predictions, we sought an approach that would relate the different types of nearest-neighbor interactions, thereby constraining the model's parameters and improving its predictive power. We first assumed that cooperative interactions are transmitted along the thin filament solely through tropomyosin (Tm) and its linkages with neighboring Tm molecules. We adopted published values from structural studies for the azimuthal angle displacements of Tm as it transitions between blocked, closed, and open regulatory states. We then postulated a mechanical model by which nearest-neighbor Tms alter the activation energy of tropomyosin state transitions according to structurally motivated strain energy functions. Making simple assumptions about the functional form of the strain energy then enables kinetic rates to depend explicitly on the characteristic stiffness of the Tm chain. Our formulation naturally results in multiple forms of cooperativity that are interrelated in a compact, structurally motivated manner while reducing the number of free parameters. The model successfully fits measured steady state and dynamic Ca2+-force relations, and preliminary simulations indicate that varying Tm stiffness has complex effects on thin filament activation. This behavior suggests that Tm-Tm coupling provides both inhibiting and activating cooperation between RUs, and that these effects are non-monotonically dependent on the magnitude of Tm stiffness.

Contributions of Ca2+-Independent Thin Filament Activation to Cardiac Muscle Function

Although Ca2+ is the principal regulator of contraction in striated muscle, in vitro evidence suggests that some actin-myosin interaction is still possible even in its absence. Whether this Ca2+-independent activation (CIA) occurs under physiological conditions remains unclear, as does its potential impact on the function of intact cardiac muscle. The purpose of this study was to investigate CIA using computational analysis. We added a structurally motivated representation of this phenomenon to an existing myofilament model, which allowed predictions of CIA-dependent muscle behavior. We found that a certain amount of CIA was essential for the model to reproduce reported effects of nonfunctional troponin C on myofilament force generation. Consequently, those data enabled estimation of ΔGCIA, the energy barrier for activating a thin filament regulatory unit in the absence of Ca2+. Using this estimate of ΔGCIA as a point of reference (∼7 kJ mol−1), we examined its impact on various aspects of muscle function through additional simulations. CIA decreased the Hill coefficient of steady-state force while increasing myofilament Ca2+ sensitivity. At the same time, CIA had minimal effect on the rate of force redevelopment after slack/restretch. Simulations of twitch tension show that the presence of CIA increases peak tension while profoundly delaying relaxation. We tested the model’s ability to represent perturbations to the Ca2+ regulatory mechanism by analyzing twitch records measured in transgenic mice expressing a cardiac troponin I mutation (R145G). The effects of the mutation on twitch dynamics were fully reproduced by a single parameter change, namely lowering ΔGCIA by 2.3 kJ mol−1 relative to its wild-type value. Our analyses suggest that CIA is present in cardiac muscle under normal conditions and that its modulation by gene mutations or other factors can alter both systolic and diastolic function.

Truncation of the Mobile Domain of Cardiac Troponin I Results in Biphasic Calcium-Dependent Thin Filament Activation

Cardiac troponin I (cTnI) contains a ∼40 residue C-terminal mobile domain (cTnI-Md, residues 168-211 in Rattus norvegicus) that is known to promote the blocked state of thin filament regulation. Because it has also been shown to stabilize the closed-state position of tropomyosin, we hypothesized that cTnI-Md is involved in the cooperativity of thin filament activation. To test this hypothesis, we generated the truncation mutant cTnI(1-167) from R.norvegicus wherein the entire cTnI-Md had been removed. We used passive exchange to incorporate cTnI(1-167) and FRET labeled cTnC(T13C/N51C)AEDENS-DDPM into left ventricular detergent skinned myocardial fibers. SDS-PAGE and Western blotting demonstrated that cTnC(T13C/N51C)AEDENS-DDPM and cTnI(1-167) were efficiently exchanged into skinned fibers. Intriguingly, incorporation of cTnI(1-167) resulted in biphasic Ca2+ dependent thin filament activation as indicated by the tightly coupled force−Ca2+ and N-cTnC-opening-Ca2+ relationships. Simultaneous force and FRET measurements showed that treatment with 1 mM orthovanadate inhibited force, reduced ensemble-averaged N-cTnC opening, and decreased the Ca2+-sensitivity of activation, but did not affect the cooperativity underlying the biphasic response of N-cTnC opening to increasing Ca2+. Akaike information criteria indicated that a weighted sum of two Hill equations was >109-fold superior in describing biphasic activation than a single Hill equation. Interestingly, a steady-state cooperativity model based on the concept of tropomyosin being pinned by cTnI (J. Mol. Biol., vol. 340, pp. 295-305) was 41-fold superior to the weighted sum of two Hill equations and suggested that blocked-state allosteric communication is severely disrupted by removal of cTnI-Md. We concluded that cTnI-Md helps facilitate allosteric communication between thin filament regulatory units in the blocked state and is therefore essential to achieving a proper contractile response to sarcomeric Ca2+ signals during early systole.

The troponin I: inhibitory peptide uncouples force generation and the cooperativity of contractile activation in mammalian skeletal muscle

Hodges and his colleagues identified a 12 amino acid fragment of troponin I (TnI-ip) that inhibits Ca(2+)-activated force and reduces the effectiveness Ca(2+) as an activator. To understand the role of troponin C (TnC) in the extended cooperative interactions of thin filament activation, we compared the effect of TnI-ip with that of partial troponin TnC extraction. Both methods reduce maximal Ca(2+)-activated force and increase [Ca(2+)] required for activation. In contrast to TnC extraction, TnI-ip does not reduce the extended cooperative interactions between adjacent thin filament regulatory units as assessed by the slope of the pCa/force relationship. Additional evidence that TnI-ip does not interfere with extended cooperativity comes from studies that activate muscle by rigor crossbridges (RXBs). TnI-ip increases both the cooperativity of activation and the concentration of RXBs needed for maximal force. This shows that TnI-ip binding to TnC increases the stability of the relaxed state of the thin filament. TnI-ip, therefore, uncouples force generation from extended cooperativity in both Ca(2+) and RXB activated muscle contraction. Because maximum force can be reduced with no change-or even an increase-in cooperativity, force-generating crossbridges do not appear to be the primary activators of cooperativity between thin filament regulatory units of skeletal muscle.

Slowed Dynamics of Thin Filament Regulatory Units Reduces Ca2+-Sensitivity of Cardiac Biomechanical Function

Actomyosin kinetics in both skinned skeletal muscle fibers at maximum Ca2+-activation and unregulated in vitro motility assays are modulated by solvent microviscosity in a manner consistent with a diffusion limited process. Viscosity might also influence cardiac thin filament Ca2+-regulatory protein dynamics. In vitro motility assays were conducted using thin filaments reconstituted with recombinant human cardiac troponin and tropomyosin; solvent microviscosity was varied by addition of sucrose or glucose. At saturating Ca2+, filament sliding speed (s) was inversely proportional to viscosity. Ca2+-sensitivity (pCa50 ) of s decreased markedly with elevated viscosity (η/η0 ≥ ~1.3). For comparison with unloaded motility assays, steady-state isometric force (F) and kinetics of isometric tension redevelopment (kTR ) were measured in single, permeabilized porcine cardiomyocytes when viscosity surrounding the myofilaments was altered. Maximum Ca2+-activated F changed little for sucrose ≤ 0.3 M (η/η0 ~1.4) or glucose ≤ 0.875 M (η/η0 ~1.66), but decreased at higher concentrations. Sucrose (0.3 M) or glucose (0.875 M) decreased pCa50 for F. kTR at saturating Ca2+ decreased steeply and monotonically with increased viscosity but there was little effect on kTR at sub-maximum Ca2+. Modeling indicates that increased solutes affect dynamics of cardiac muscle Ca2+-regulatory proteins to a much greater extent than actomyosin cross-bridge cycling.

Filament Compliance Influences Cooperative Activation of Thin Filaments and the Dynamics of Force Production in Skeletal Muscle

Striated muscle contraction is a highly cooperative process initiated by Ca2+ binding to the troponin complex, which leads to tropomyosin movement and myosin cross-bridge (XB) formation along thin filaments. Experimental and computational studies suggest skeletal muscle fiber activation is greatly augmented by cooperative interactions between neighboring thin filament regulatory units (RU-RU cooperativity; 1 RU = 7 actin monomers+1 troponin complex+1 tropomyosin molecule). XB binding can also amplify thin filament activation through interactions with RUs (XB-RU cooperativity). Because these interactions occur with a temporal order, they can be considered kinetic forms of cooperativity. Our previous spatially-explicit models illustrated that mechanical forms of cooperativity also exist, arising from XB-induced XB binding (XB-XB cooperativity). These mechanical and kinetic forms of cooperativity are likely coordinated during muscle contraction, but the relative contribution from each of these mechanisms is difficult to separate experimentally. To investigate these contributions we built a multi-filament model of the half sarcomere, allowing RU activation kinetics to vary with the state of neighboring RUs or XBs. Simulations suggest Ca2+ binding to troponin activates a thin filament distance spanning 9 to 11 actins and coupled RU-RU interactions dominate the cooperative force response in skeletal muscle, consistent with measurements from rabbit psoas fibers. XB binding was critical for stabilizing thin filament activation, particularly at submaximal Ca2+ levels, even though XB-RU cooperativity amplified force less than RU-RU cooperativity. Similar to previous studies, XB-XB cooperativity scaled inversely with lattice stiffness, leading to slower rates of force development as stiffness decreased. Including RU-RU and XB-RU cooperativity in this model resulted in the novel prediction that the force-[Ca2+] relationship can vary due to filament and XB compliance. Simulations also suggest kinetic forms of cooperativity occur rapidly and dominate early to get activation, while mechanical forms of cooperativity act more slowly, augmenting XB binding as force continues to develop.

Regulation of Fibre Contraction in a Rat Model of Myocardial Ischemia

BackgroundThe changes in the actomyosin crossbridge cycle underlying altered contractility of the heart are not well described, despite their importance to devising rational treatment approaches.Methodology/Principal FindingsA rat ischemia–reperfusion model was used to determine the transitions of the crossbridge cycle impacted during ischemia. Compared to perfused hearts, the maximum force per cross-sectional area and Ca2+ sensitivity of fibers from ischemic hearts were both reduced. Muscle activation by photolytic release of Ca2+ and ATP suggested that the altered contractility was best described as a reduction in the rate of activation of noncycling actomyosin crossbridges to activated, cycling states. More specifically, the apparent forward rate constant of the transition between the nonforce bearing A-M.ADP.Pi state and the bound, force bearing AM*.ADP.Pi state was reduced in ischemic fibers, suggesting that this transition is commensurate with initial crossbridge activation. These results suggested an alteration in the relationship between the activation of thin filament regulatory units and initial crossbridge attachment, prompting an examination of the post-translational state of troponin (Tn) T and I. These analyses indicated a reduction in the diphosphorylated form of TnT during ischemia, along with lower Ser23/24 phosphorylation of TnI. Treatment of perfused fibers by 8-Br-cAMP increased Ser23/24 phosphorylation of TnI, altering the reverse rate constant of the Pi isomerization in a manner consistent with the lusitropic effect of β-adrenergic stimulation. However, similar treatment of ischemic fibers did not change TnI phosphorylation or the kinetics of the Pi isomerization.ConclusionsIschemia reduces the isomerization from A-M.ADP.Pi to AM*.ADP.Pi, altering the kinetics of crossbridge activation through a mechanism that may be mediated by altered TnT and TnI phosphorylation.

The Combined Effects of ADP, ATP, and Myosin Density on Cooperative Activation of Thin Filaments

Cooperative activation of thin filaments has been achieved by decreasing [ATP], increasing [ADP], and increasing myosin densities; however, these observations have yet to be incorporated into a self-consistent description of thin filament regulation. It has been noted in previous skinned muscle fiber studies that contraction can be initiated with high [ADP] when Ca2+ is absent. Similarly, low [ATP] has been shown to activate thin filaments in Ca2+-free motility assays. Based on these observations, we hypothesize that high concentrations of ADP can cause thin filament cooperative activation in the absence of Ca2+. Using an in vitro motility assay, we determine the effects of ATP, ADP, and myosin density on the cooperative activation of myosin-based thin filament sliding, V. At 1mM [ATP], V is activated over an [ADP] range from ∼2 to 8mM and becomes increasingly inhibited at [ADP] >8mM. This biphasic effect of [ADP] on V is similar to the effects of [ATP] on cooperative thin filament activation previously measured in vitro. The observed effects of [ADP] and [ATP] on V are consistent with a model in which increasing [ADP] or decreasing [ATP] increase the probability that a myosin head is bound within a thin filament regulatory unit, cooperatively activating that regulatory unit (and possible adjacent ones) and increasing V. At sufficiently high [ADP] or sufficiently low [ATP], the thin filament becomes fully activated, and a further increase in [ADP] or decrease in [ATP] slows V by inhibiting detachment kinetics.

Tropomyosin Phosphorylation Has Filament-Level And Crossbridge-Level Effects On Actin-Myosin Interactions

The α-isoform of tropomyosin (Tm) can be phosphorylated near the C-terminus, and dephosphorylation of Tm has been shown to decrease actin-myosin ATPase rates (Heeley et al., 1989). Additionally, Tm polymerizes with adjacent Tm molecules in a head-to-tail manner. Phosphorylation of Tm might therefore influence the strength of these interactions, and thus regulate the degree of cooperative activation by myosin binding. We tested Tm phosphorylation effects by measuring the force and velocity of actin-myosin interactions at the level of single actin filaments using a combination of actin-Tm binding assays, in vitro motility assays, and a novel, high throughput laser trap assay to measure isometric force. In these assays we used purified heavy meromyosin (HMM) and actin filaments reconstituted with either natively phosphorylated or dephosphorylated Tm. Dephosphorylation of Tm did not result in significant changes in actin-Tm binding or unloaded sliding velocities. Isometric force measurements showed that thin filaments were cooperatively activated by myosin, but only when Tm was phosphorylated. When Tm was dephosphorylated, the reconstituted filaments behaved like bare actin filaments. These results suggest transmission of cooperative activation beyond one thin filament regulatory unit when Tm is phosphorylated. Moreover, Tm phosphorylation increased isometric force production ∼50% compared to bare actin filaments at intermediate HMM surface densities. In combination with sliding velocity data, this result suggests that Tm phosphorylation may have effects at the level of a single crossbridge that cannot be explained by steric hindrance. We hypothesize that Tm phosphorylation accelerates the rate of crossbridge attachment. Single molecule kinetic and force spectroscopy experiments are underway to gain insight on this possibility.

Role of Strongly-Bound Crossbridges in Cooperative Cardiac Thin Filament Activation

Experimental evidence indicates that activation of cardiac thin filaments is enhanced by strongly-bound myosin crossbridges and that crossbridge binding is in turn cooperatively regulated by end-to-end interactions between adjacent tropomyosins. We examined the impact of crossbridge binding and nearest-neighbor tropomyosin interactions on thin filament activation using a computational model.We represented individual thin filament regulatory units (RUs) with the model of McKillop and Geeves [Biophys J, 1993 65(2)] wherein RUs are found in blocked (B, non-permissive), closed (C, permissive), and open (M, permissive with crossbridge) states. The B to C transition was assumed to depend upon Ca2+ concentration. Nearest-neighbor RU interactions were represented by causing transitions of the individual RU model to depend on the status of neighbors. Ensembles of N interacting RUs were modeled as Markov networks generated by considering all possible unique configurations of individual RUs (B, C, or M) within a chain.The model-generated steady-state force-pCa curve (N=6) possessed a Hill coefficient of 3.0. Hill coefficients fit separately to portions of the curve below and above half activation were 3.2 and 2.4, respectively. Rate of force redevelopment following rapid slack/restretch (ktr) showed strong dependence on activation level (ktr=2.6 s-1 at pCa 6.0 vs. 9.3 s-1 at pCa 4.3). Increasing the crossbridge duty cycle in the model increased myofilament Ca2+ sensitivity but had an opposite effect on Ca2+ sensitivity of ktr. Simultaneous matching of reported force and ktr sensitivities required a duty cycle of 30%. Increasing N toward 26 (a realistic filament length) tended to improve the fit with experiments.These results suggest that cycling crossbridges act through nearest-neighbor interactions along the thin filament to 1) increase myofilament Ca2+ sensitivity, 2) cooperatively enhance activation, and 3) slow the rate of force redevelopment at low levels of activation.

Sarcomere Length Dependence Of The Force-pCa Relationship In Cardiac Muscle Is Influenced By Properties Of Thin Filament Regulatory Units

Myocardial performance is tightly regulated by sarcomere length (SL), which is thought to be due at least in part to concurrent changes in myofilament lattice spacing and the relative proximity (and binding probability) of myosin heads to actin. Furthermore, crossbridge binding enhances Ca2+ binding to troponin (Tn) in cardiac muscle, a unique form of cooperative thin filament activation whose mechanism is currently unknown. Experiments here were designed to test the hypothesis that the properties of Tn are important in determining the SL dependence of force in cardiac muscle. We compared trabeculae exchanged with WT cTn vs. cTn containing a mutant (L48Q) cTnC with enhanced Ca2+ affinity. L48Q cTnC caused a left-shift in the force-pCa relationship at long (2.3 μm) SL as compared to WT cTnC. Interestingly, L48Q cTnC effectively eliminated SL-dependence of the force-pCa relationship, via a much larger left-shift at short SL, while SL dependence of Fmax was unaffected. This suggests that SL-dependence of cardiac force development can be greatly influenced by the properties of native cTn, perhaps by limiting crossbridge binding, and that this effect is likely most important at shorter SL. Furthermore, increasing the Ca2+ binding and/or cTnC-cTnI interaction properties of cTn (such as with L48Q cTnC) can reduce or eliminate this limitation. Ongoing experiments will determine whether TnI phosphorylation can restore SL dependence by decreasing Tn Ca2+ affinity, thereby reducing myosin access to actin binding sites. This will also be tested by exchange with I61Q cTnC-Tn, a mutant with reduced Ca2+ affinity. Overall, these results imply that the cardiac length-force relationship is governed, at least in part, by properties of thin filament regulatory units. Support appreciated from NIH R01 HL 65497 (MR) and T32 HL07828 (FSK).

Influence of enhanced troponin C Ca2+‐binding affinity on cooperative thin filament activation in rabbit skeletal muscle

We studied how enhanced skeletal troponin C (sTnC) Ca2+-binding affinity affects cooperative thin filament activation and contraction in single demembranated rabbit psoas fibres. Three sTnC mutants were created and incorporated into skeletal troponin (sTn) for measurement of Ca2+ dissociation, resulting in the following order of rates: wild-type (WT) sTnC-sTn>sTnC(F27W)-sTn>M80Q sTnC-sTn>M80Q sTnCF27W-sTn. Reconstitution of sTnC-extracted fibres increased Ca2+ sensitivity of steady-state force (pCa(50)) by 0.08 for M80Q sTnC, 0.15 for sTnCF27W and 0.32 for M80Q sTnCF27W with minimal loss of slope (nH, degree of cooperativity). Near-neighbour thin filament regulatory unit (RU) interactions were reduced in fibres by incorporating mixtures of WT or mutant sTnC and D28A, D64A sTnC (xxsTnC) that does not bind Ca2+ at N-terminal sites. Reconstitution with sTnC: xxsTnC mixtures to 20% of pre-exchanged maximal force reduced pCa50 by 0.35 for sTnC: xxsTnC, 0.25 for M80Q sTnC: xxsTnC, and 0.10 for M80Q sTnCF27W: xxsTnC. It is interesting that pCa50 increased by approximately 0.1 for M80Q sTnC and approximately 0.3 for M80Q sTnCF27W when near-neighbour RU interactions were reduced; these values are similar in magnitude to those for fibres reconstituted with 100% mutant sTnC. After reconstitution with sTnC: xxsTnC mixtures, nH decreased to a similar value for all mutant sTnCs. Altered sTnC Ca2+-binding properties (M80Q sTnCF27W) did not affect strong crossbridge inhibition by 2,3-butanedione monoxime when near-neighbour thin filament RU interactions were reduced. Together these results suggest increased sTnC Ca2+ affinity strongly influences Ca2+ sensitivity of steady-state force without affecting near-neighbour thin filament RU cooperative activation or the relative contribution of crossbridges versus Ca2+ to thin filament activation.

Thin‐filament regulation of force redevelopment kinetics in rabbit skeletal muscle fibres

Thin-filament regulation of isometric force redevelopment (k(tr)) was examined in rabbit psoas fibres by substituting native TnC with either cardiac TnC (cTnC), a site I-inactive skeletal TnC mutant (xsTnC), or mixtures of native purified skeletal TnC (sTnC) and a site I- and II-inactive skeletal TnC mutant (xxsTnC). Reconstituted maximal Ca(2+)-activated force (rF(max)) decreased as the fraction of sTnC in sTnC: xxsTnC mixtures was reduced, but maximal k(tr) was unaffected until rF(max) was <0.2 of pre-extracted F(max). In contrast, reconstitution with cTnC or xsTnC reduced maximal k(tr) to 0.48 and 0.44 of control (P < 0.01), respectively, with corresponding rF(max) of 0.68 +/- 0.03 and 0.25 +/- 0.02 F(max). The k(tr)-pCa relation of fibres containing sTnC: xxsTnC mixtures (rF(max) > 0.2 F(max)) was little effected, though k(tr) was slightly elevated at low Ca(2+) activation. The magnitude of the Ca(2+)-dependent increase in k(tr) was greatly reduced following cTnC or xsTnC reconstitution because k(tr) at low levels of Ca(2+) was elevated and maximal k(tr) was reduced. Solution Ca(2+) dissociation rates (k(off)) from whole Tn complexes containing sTnC (26 +/- 0.1 s(-1)), cTnC (38 +/- 0.9 s(-1)) and xsTnC (50 +/- 1.2 s(-1)) correlated with k(tr) at low Ca(2+) levels and were inversely related to rF(max). At low Ca(2+) activation, k(tr) was similarly elevated in cTnC-reconstituted fibres with ATP or when cross-bridge cycling rate was increased with 2-deoxy-ATP. Our results and model simulations indicate little or no requirement for cooperative interactions between thin-filament regulatory units in modulating k(tr) at any [Ca(2+)] and suggest Ca(2+) activation properties of individual troponin complexes may influence the apparent rate constant of cross-bridge detachment.

Functional consequence of mutation in rat cardiac troponin T is affected differently by myosin heavy chain isoforms

Cardiac troponin T (cTnT) is an essential component of the thin filament regulatory unit (RU) that regulates Ca2+ activation of tension in the heart muscle. Because there is coupling between the RU and myosin crossbridges, the functional outcome of cardiomyopathy-related mutations in cTnT may be modified by the type of myosin heavy chain (MHC) isoform. Ca2+ activation of tension and ATPase activity were measured in muscle fibres from normal rat hearts containing alpha-MHC isoform and propylthiouracil (PTU)-treated rat hearts containing beta-MHC isoform. Muscle fibres from normal and PTU-treated rat hearts were reconstituted with two different mutations in rat cTnT; the deletion of Glu162 (cTnT(E162DEL)) and the deletion of Lys211 (cTnT(K211DEL)). Alpha-MHC and beta-MHC isoforms had contrasting impact on tension-dependent ATP consumption (tension cost) in cTnT(E162DEL) and cTnT(K211DEL) reconstituted muscle fibres. Significant increases in tension cost in alpha-MHC-containing muscle fibres corresponded to 17% (P < 0.01) and 23% (P < 0.001) when reconstituted with cTnT(E162DEL) and cTnT(K211DEL), respectively. In contrast, tension cost decreased when these two cTnT mutants were reconstituted in muscle fibres containing beta-MHC; by approximately 24% (P < 0.05) when reconstituted with cTnT(E162DEL) and by approximately 17% (P = 0.09) when reconstituted with cTnT(K211DEL). Such differences in tension cost were substantiated by the mechano-dynamic analysis of cTnT mutant reconstituted muscle fibres from normal and PTU-treated rat hearts. Our observation demonstrates that qualitative changes in MHC isoform alters the nature of cardiac myofilament dysfunction induced by mutations in cTnT.

Physiological consequences of thin filament cooperativity for vertebrate striated muscle contraction: a theoretical study

Bindings of both myosin and Ca(2+) to the thin filament of vertebrate striated muscle are known to be strongly cooperative. Here the relation between these two sources of cooperativity and their consequences for physiological properties are assessed by comparing two models, with and without Monod-type myosin-binding cooperativity. In both models a thin filament regulatory unit (RU) is in either 'off' or 'on' state, and the equilibrium between them (K (on)) is [Ca(2+)]-dependent. The calculations predict the following: (1) In both models, myosin binding stabilizes the RU in the 'on' state, causing troponin to trap Ca(2+). This stabilization in turn increases the Ca(2+)-binding cooperativity, ensuring efficient regulation to occur in a narrow [Ca(2+)] range. (2) In the cooperative model, the RU is stabilized with a relatively low myosin affinity for actin (K approximately approximately 1), while the non-cooperative model requires a much higher affinity (K approximately approximately 10) to produce the same effect. (3) The cooperative model reproduces the known effects of [Ca(2+)] on the rate of force development and shortening velocity with a low K, but again the non-cooperative model requires a higher value. (4) Because of the finite value of K (on), the thin filaments can never be fully activated by increasing [Ca(2+)], indicating that contracting muscles are under strong influence of thin-filament cooperativity even at saturating [Ca(2+)]. Interpretation of data on muscle mechanics without considering these cooperative effects could therefore lead to a substantial (10-fold) overestimate of cross-bridge binding properties.